Traditional approaches in drug development have focused on the use of therapeutic agents capable of interacting directly with proteins involved in disease states or other states of unhealth. Drugs borne of this tradition include, for example, synthetic hormones (to simulate the function of protein-based hormones desirably present in the body), antibiotics (which attack foreign proteins, namely those of microorganisms) and vitamins (which provide the building blocks required by certain proteins to perform their ordinary function in the body), in addition to many others. More recently, therapeutic agents in the form of oligonucleotides have been designed to indirectly regulate, control, or otherwise impact protein function by altering at the genetic level the blueprint or machinery that controls synthesis of all proteins. Because each gene contains the information necessary to produce many copies of a particular protein, each of these nucleic acid therapeutic agents can affect a greater number of protein molecules through its indirect interaction than can a traditional macromolecular drug that relies on direct interaction with the targeted protein.
Nucleic acid therapeutic compounds may act in a number of different ways, but will most commonly fall into either one of two categories. The first category includes oligonucleotides that simulate or potentiate in some way a desired genetic effect. The activity stimulated by this type of nucleic acid therapeutic compound is commonly referred to as "gene therapy". The second category of nucleic acid therapeutic compounds includes inhibitory oligonucleotides wherein the nucleic acid therapeutic compound inhibits the production of undesired proteins. Antisense oligonucleotides form a subclass of inhibitory nucleic acid therapeutic compounds, although compounds commonly assigned to this subclass may not always act in a true "antisense" manner. In addition to these two categories of therapeutic oligonucleotides, it should also be noted that it is also possible for nucleic acid therapeutic compounds to interact directly with the target proteins in much the same way as traditional therapeutic drugs.
True antisense interactions involve the hybridization of complementary oligonucleotides (hence, the term "antisense") to their selected nucleic acid target (e.g., viral RNA or other undesired genetic messages) in a sequence specific manner such that the complex thus formed, either alone or in combination with other reagent(s) (e.g., enzymes, such as RNAse) can no longer function as a template for the translation of genetic information into proteins. Other inhibitory oligonucleotides have sequences that are not necessarily complementary to a target sequence, but, like antisense oligonucleotides, have the potential to interfere with the expression (e.g., replication and/or translation) of the undesired genetic material. An antisense oligonucleotide may be designed to interfere with the expression of foreign genes (e.g., viral genes, such as HIV) or with the aberrant expression of endogenous genes (e.g., a normal gene that is aberrantly expressed as a mutated oncogene). These undesired genetic messages are involved in many disease states, including viral infections and carcinomas. Inhibitory oligonucleotides raise the possibility of therapeutic arrest of a disease state at the early replication and expression stage, rather than attacking the resulting protein at a later stage of disease progression as in the manner of traditional drugs.
Oligonucleotides used in gene therapy are designed to provide an oligonucleotide, or synthetic gene, having a desired effect that is otherwise absent or impaired in a patient. Each gene normally present in a human body is responsible for the manufacture of a particular protein that contributes to either the structure or functioning of the body. If this gene is defective or absent, protein synthesis will be faulty or nonexistent, and a deformity or genetic disease will result. Incorporation of nucleic acid therapeutic compounds into the genetic material of a patient's cells can be accomplished through a vehicle, such as a retrovirus, thus enabling production of the needed protein.
Irrespective of whether nucleic acid therapeutic compounds are designed for gene therapy, antisense therapy, or any other situation where it is desired to affect proteins at a genetic or other level, the design of these synthetic oligonucleotides is a key to the level of success that can be achieved. Importantly, these oligonucleotides must ordinarily be modified in a manner that imparts nuclease resistance to the oligonucleotide such that they are capable of surviving in the presence of the various nucleases that are endogenous to a human or animal body. The same holds true for oligonucleotide probes employed in the analysis of serum samples, because the same exogenous nucleases present in the human body that can degrade unmodified therapeutic oligonucleotides are also present in human serum and can degrade unmodified oligonucleotide probes in these samples as well.
Specifically, unmodified (or "wild type") oligonucleotides are susceptible to nuclease degradation at both the 3'- and 5'-positions of the inter-nucleotide bonds that link the individual nucleoside units together in the completed oligonucleotide. Consequently, attempts to impart nuclease resistance to therapeutic oligonucleotides have been directed to modification of this internucleotide linkage, with success having been achieved primarily with respect to modification of the "non-bridging" oxygen atoms in the naturally occurring phosphodiester linkage. (E.g., phosphorothioate-modified oligonucleotides having a single non-bridging oxygen substituted with a sulfur atom (U.S. Pat. No. 3,846,402) and phosphorodithioate-modified oligonucleotides having both non-bridging oxygen atoms substituted with sulfur atoms (U.S. Pat. No. 5,218,103)). It has been observed, however, that phosphorothioate-modified oligonucleotides remain susceptible to nuclease degradation at the 3'-position of the modified internucleotide bonds in some instances, especially by nucleases leaving a 5'-phosphate following cleavage of the internucleotide bond. This is presumably due to the fact that only one of the "non-bridging" oxygen atoms in the phosphodiester bond is modified.
Other attempts to impart nuclease resistance to therapeutic or diagnostic oligonucleotides have been directed to modification of the "bridging" oxygen atoms in the naturally occurring phosphodiester linkage, with some limited success having been achieved. For example, the synthesis of an oligonucleotide containing a single 3'-methylene substitution (i.e., the 3'-bridging oxygen is substituted with a methylene (--CH.sub.2 --) group) has been reported. Heinemann et al., Nucleic Acids Res., 19, 427-433 (1991). However, synthesis of the nucleoside intermediates required for the reported solution-based phosphotriester method of generating the internucleotide linkage is long and laborious, making the synthesis of multiple 3'-methylene modifications in an oligonucleotide difficult and tedious. Consequently, nuclease stability for oligonucleotides containing the 3'-methylene linkage have only recently been reported.
Modified oligonucleotides containing bridged phosphoramidate linkages (i.e., either the 5'-oxygen or the 3'-oxygen is replaced by an amino (--NH--) group) have been synthesized. Gryaznov et al., Nucleic Acids Res., 20, 3403-3409, (1992) and Mag et al., Tetrahedron Lett., 33, 7323-7326 (1992). Similarly, modified oligonucleotides containing 3'-thio-bridged linkages (3'-bridging oxygen substituted with a sulfur atom) have been synthesized. Cosstick et al., Nucleic Acids Res., 18, 829-834 (1990); Vyle et al., Biochemistry, 31, 3012-3018 (1992). Modification of the 3'-oxygen by an amino or sulfur moiety has been found to confer some resistance to nuclease degradation, however, it has been observed that phosphoramidate and 3'-thio-bridged modified oligonucleotides remain susceptible to degradation of the modified internucleotide bond, presumably because a single modification to only one of the "bridging" oxygens is insufficient to confer significant nuclease resistance (i.e., similar to the above-described phosphorothioate modification).
The synthesis of modified oligonucleotides containing a single 5'-thio-bridged substitution (5'-bridging oxygen substituted with a sulfur atom) has also been reported. Mag et al., Nucleic Acids Research, 19, 1437-1441 (1991). However, this method is not compatible with the synthesis of modified oligonucleotides having more than a single 5'-thio-bridged linkage, because the deprotection reaction required to complete formation of the desired modified internucleotide bond employs silver or mercury salts to cleave the trityl protecting group from the 5'-thiol moiety. These salts would also necessarily cleave any previously formed P--S bond in the oligonucleotide. Other known solution-phase methods for making modified oligonucleotides containing multiple 5'-thio-bridged modifications are based on phosphodiester technology and are incompatible with automated, polymer-supported synthesis of oligonucleotides. Moreover, the 5'-thio-bridged modification alone would not be expected to impart sufficient nuclease resistance to oligonucleotides for use as a nucleic acid therapeutic, because only one of the "bridging" oxygens is modified.
It would be desirable to have a further dithio-modified oligonucleotide of a length that would be suitable for use as a nucleic acid therapeutic compound or as a diagnostic probe and would have a type of thio-bridged modified linkage that is able to impart nuclease resistance to the modified oligonucleotide.
Therefore, it is an object of the present invention to provide dithio-modified oligonucleotides having a sulfur substitution at the 5'-position of at least one of the internucleotide linkages.
It is a further object of the present invention to provide a polymer-supported method for synthesis of oligonucleotides having a 5'-bridging sulfur substitution of one or more of the internucleotide bonds.
It is a still further object of the present invention to provide novel nucleoside intermediates for use in the synthesis of oligonucleotides having 5'-thio-bridged modification(s).